Journal:  Journal of Medical Microbiology

Title: Quantification of cholix exotoxin, an ADP-ribosylating factor in Vibrio cholerae strains by developed sandwich bead-ELISA.

Authors:  Sharda Prasad Awasthi, Nityananda Chowdhury, Noritoshi Hatanaka, Atsushi Hinenoya, Thandavarayan Ramamurthy, Masahiro Asakura and Shinji Yamasaki*

Abstract:
Introduction. Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced by Vibrio cholerae. However, to date, there is no quantitative assay available for ChxA, which makes it difficult to detect and estimate the level of ChxA produced by V. cholerae.
Hypothesis/Gap Statement. It is important to develop a reliable and specific quantitative assay to measure the production level of ChxA, which will help us to understand the role of ChxA in V. cholerae pathogenesis.
Aim. The aim of this study was to develop a bead-based sandwich ELISA (bead-ELISA) for the quantification of ChxA and to evaluate the importance of ChxA in the pathogenesis of V. cholerae infection.
Methodology. Anti-rChxA was raised in New Zealand white rabbits, and Fab-horse radish peroxidase conjugate was prepared by the maleimide method to use in the bead-ELISA. This anti-ChxA bead-ELISA was applied to quantify the ChxA produced by various V. cholerae strains. The production of ChxA was examined in different growth media such as alkaline peptone water (APW), Luria-Bertani broth and AKI. Finally, the assay was evaluated using a mouse lethality assay with representative V. cholerae strains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA.
Results. A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng/ml of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng/ml to 1.6 µg/ml. The highest ChxA production was observed when V. cholerae strains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producer V. cholerae strains showed 20–80 % lethality and only the high ChxA II-producer was statistically more lethal than a non-ChxA-producer, in the mice model assay. ChxA I and II production levels were not well correlated with mice lethality, and this could be due to the heterogeneity of the strains tested.
Conclusion. ChxA I to III was produced mostly extracellularly at various levels depending on strains and culture conditions. The bead-ELISA developed in this study is useful for the detection and quantification of ChxA in V. cholerae strains.

This study, for the first time, developed a cholix toxin-specific sandwich bead-ELISA by which the toxin production of V. cholerae can be quantitatively measured.